To understand intracellular protein-protein and protein-nucleic acid interactions, it is critical to appreciate their spatial relationships, to assess their local concentrations in cells, and to provide this information dynamically over a time course of minutes to hours.
Computed tomography (CT) imaging of freely suspended particles, including live single cells and cell clusters, is made possible by recent developments in low-light level imaging and other detectors, microelectronics, microfluidics and high-speed computing. By studying live single cells in their microenvironments, information about particularly interesting cell lines and their phenotypes can be obtained. Single cell analysis (SCA) methods are finding increasing use for studying disease progression, development, treatment, and prognosis because conventional population based measurement techniques often mask important heterogeneous responses that are inherent to the disease state. Heterogeneity among cell populations plays an important role in diseases like cancer, and in resistance to its treatment. Therefore, SCA is recognized as an important field of study.
Microfluidic systems possess some inherent unique and advantageous attributes due to the physics of scale. For instance, they generally have low Reynolds numbers, which assures laminar flow conditions, and mass transport is diffusion dominated, providing significant advantages for mixing and dispersion, especially for live cells.
Various forces (e.g., electrical, optical, mechanical, magnetic, chemical, and thermal) have been used for single cell analysis and manipulation. Mechanical methods have been utilized for single cell and microparticle rotation using the concept of a microvortex; which relies on the creation of a recirculating flow profile. (Shelby J P, Chiu D T. “Controlled rotation of biological micro- and nano-particles in microvortices.” Lab Chip. 2004; 4(3):168-70.) The center of the microvortex is the site of a trapping force, which is locally induced by the flow velocity gradient in that region. Rotation of a cell 1 about an axis 2 parallel to the optical axis 3 (e.g., 2D rotation) is schematically illustrated in FIG. 1A. One way of rotating single cells about an axis parallel to the optical axis using microvortices is to fabricate channels with diamond shaped side chambers, thus allowing flow to be peeled off from the main channel and become recirculant in the side channel, thereby forming a microvortex. (Lim D S W, Shelby J P, Kuo J S, Chiu D T. “Dynamic formation of ring-shaped patterns of colloidal particles in microfluidic systems.” Appl. Phys. Lett. 2003; 83(6):1145.) The dimensions of the opening chamber and especially its opening angle and aspect ratio are important factors that affect stability of the microvortex. To exert better control on the cells during rotation, an optical trap may be used to position the cell at the center of the microvortex. (Neuman K C, Block S M. “Optical trapping.” Rev. Sci. Instrum. 2004; 75(9):2787-809.)
Many diseases arise from genomic changes, some of which manifest at the cellular level in cytostructural and protein expression features which can be resolved, captured, and quantified in 3D-microscopy far more sensitively and specifically than in traditional 2D-microscopy. Volumetric cell imaging using 3D optical Computed Tomography (cell CT) would be advantageous for identification and characterization of various cells, including cancer cells. To perform 3D imaging, it is necessary to have the ability to hold microscopic particles precisely in free suspension and to slowly rotate them. Rotation of a cell 1 about an axis 4 perpendicular to the optical axis 3 (e.g., 3D rotation, to permit 3D imaging) is schematically illustrated in FIG. 1B. It can be tricky to prepare devices capable of 3D rotation of one or more cells to enable 3D microscopy such as live-cell or cell cluster computer tomography imaging. Aspects of this disclosure address shortcomings associated with conventional systems and methods.